Previously, antisera against recombinant outer capsid rotavirus VP4 proteins were used to study the VP4 antigenic polymorphism among human rotavirus strains. Three distinct serotypes and one subtype of the VP4 protein were identified among 17 human rotavirus strains that contained representatives of five different VP7 serotypes. Immunization of mice with the VP8 cleavage subunit of VP4 of the human rotavirus KU, DS-1, 1076, or K8 strain induced antibodies that neutralized the rotavirus from which the VP4 subunit was derived. In one fusion with the spleen of a mouse inoculated with VP8-DS-1, 14 hybridomas were detected that reacted with rotavirus by ELISA. Some mAbs only recognized the homologous human rotavirus DS-1 strain, whereas others were cross-reactive. These results suggest that the VP8 subunit of VP4 contains specific regions which can be useful in distinguishing VP4 serotypes among human rotaviruses.